We propose to continue our studies on the structure and assembly of yeast cytochrome c oxidase, a transmembranous oligomeric enzyme which spans the inner mitochondrial membrane and which is a useful model system for examining the molecular events which underly the assembly of biological membranes. This enzyme is a relatively simple, well-defined, and genetically-manipulatable protein which possesses many of those properties which are basic to all biological membranes; including the possession of polypeptide subunits which are analogous to "integral" and "peripheral" membrane proteins, the possession of functional and structural asymmetry in situ, and a close association with phospholipids. Yeast cytochrome c oxidase is also ideally suited for studying the cooperative interaction between mitochondrial and nuclear genomes since it is composed of polypeptide subunits contributed by both genetic systems. In this study we propose to: 1) further characterize the newly-discovered precursors to the mitochondrially- and cytoplasmically-translated subunits; 2) identify the membrane-bound intermediates and protease along the proteolytic cleavage pathways of these precursors; 3) determine the order with which subunits, precursors, proteolytic cleavage intermediates, and phospholipids interact during the assembly of the holoenzyme; and 4) ascertain the topological relationships which subunits bear to one aother and to the "captive" lipid in the fully-assembled holoenzyme. In addition, we plan to complement these biochemical studies with genetic studies aimed at: 1) understanding the organization and regulation of the structural genes for the nuclear-encoded subunits; 2) identifying nuclear genes whose gene products are required for the proper assembly of the holoenzyme; and 3) identifying nuclear genes whose products play (a) specific role(s) in the expression of the structural genes for the mitochondrial subunits.